The following antibodies were used for western blotting: rat anti-HA (3F10; Roche/Sigma-Aldrich), rabbit anti-myc, mouse anti-RhoA, rabbit anti-MLC2 (Santa Cruz Technology), rabbit anti-FAM40B (Sigma-Aldrich), rabbit anti-pThr18/pSer19-MLC2 (Cell Signaling), mouse anti-GADPH (Millipore), secondary HRP-conjugated sheep anti-mouse IgG and donkey anti-rabbit IgG (GE Healthcare). Antibodies for immunofluorescence analysis were: rabbit anti-HA (Santa Cruz Technology), rabbit anti-pSer19-MLC2 (Cell Signaling), mouse anti-VE-cadherin (BD Biosciences), rabbit anti-ZO-1 (#61–7300; ThermoFisher Scientific), secondary AlexaFluor-488 and -647-conjugated antibodies (Molecular Probes). Cells were also stained with DAPI (DNA) and AlexaFluor-546-labeled phalloidin (F-actin; Molecular Probes).
Cell culture and siRNA transfection
Pooled human umbilical vein endothelial cells (HUVECs) obtained from Lonza or PromoCell were cultured in EBM-2 medium containing the appropriate growth factors (EGM-2) and supplemented with 2% foetal bovine serum (FBS). Cell culture dishes were coated with 10 μg/ml fibronectin for 1 h at 37 °C prior to cell seeding. HUVECs were used until passage 4. Where indicated, they were stimulated with 1 U/ml thrombin (Sigma-Aldrich) for 10 min.
COS7 cells were cultured in DMEM containing 10% FBS, penicillin (100 IU/ml) and streptomycin (100 μg/ml).
HUVECs were transfected with 50 nM siRNAs using Oligofectamine in EGM-2 medium containing 4% FCS. After 16 h, medium was changed to EGM-2 containing 2% FCS. Cells were analysed 72 h after siRNA transfection. The following siRNA sequences were obtained from Dharmacon (GE Healthcare): siFAM40A-1 (5’-GCUGAUGACUCUCGAGAAG-3′), siFAM40A-2 (5’-CAGCACAAGUACACGUCGA-3′), siFAM40B-1 (5’-GAAGGCAACUCCUCACUAA-3′), siFAM40B-2 (5’-UGACUGGGCUUACGGGAAU-3′), siControl (5’-UGGUUUACAUGUCGACUAA-3′). CCM3 siRNAs were obtained from Ambion (ThermoFisher Scientific): siCCM3–1 (5’-GUGAUACUCUGAAAACGUA-3′), siCCM3–2 (5’-AGAAAAUCCAGGUCUCACA-3′).
cDNA cloning and plasmid transfection
Human FAM40A, FAM40B and CCM3 cDNAs were kindly provided in pENTRY by Dr. Stefan Wiemann (DKFZ, Germany). The FAM40A and FAM40B cDNAs were cloned into pDEST-HA, and CCM3 into pDEST-myc, using a Gateway™ LR Clonase II Enzyme kit (Invitrogen) as recommended by the manufacturer.
HUVECs were transfected with plasmid DNA with an Amaxa Nucleofector (Lonza) according to the manufacturer’s instructions. After nucleofection, cells were plated onto fibronectin-coated glass coverslips for immunofluorescence analysis.
COS7 cells were transfected with plasmid DNA using a Genepulser II System (Bio-Rad) at 250 V, 975 μF in electroporation buffer (120 mM KCl, 10 mM K2PO4/KHPO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM MgCl2, 0.5% Ficoll 400). After 48 h, cells were lysed for immunoprecipitation analysis.
Immunoprecipitation and immunoblotting
Cells were lysed in either IP lysis buffer (20 mM Tris-Cl pH 8, 130 mM NaCl, 1% Triton X-100, 1 mM DTT, 10 mM NaF, EDTA-free protease inhibitor cocktail (Roche Applied Science), phosphatase inhibitor cocktail (Calbiochem)) or RIPA buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, 125 mM NaF, EDTA-free protease inhibitor cocktail, phosphatase inhibitor cocktail). For immunoprecipitations, COS7 cell lysates were pre-cleared by incubation with non-immune IgG agarose beads (Sigma-Aldrich) for 1 h at 4 °C, then incubated with mouse anti-HA agarose beads (Sigma-Aldrich) for 3 h at 4 °C. Beads were washed with high salt IP lysis buffer (containing 250 mM NaCl). Proteins were eluted from the beads with sample buffer (4% SDS, 160 mM Tris-Cl pH 6.8, 20% glycerol, 10 mM DTT). Empty vector was transfected as a control to demonstrate that the anti-HA beads did not pull down myc-CCM3 directly.
For pMLC2 analysis, HUVECs were lysed with Laemmli lysis buffer (80 mM Tris-Cl pH 7.5, 10% glycerol, 2% SDS, 10 mM glycerol phosphate, 1 mM Na3VO4, 1 mM DTT, 10 mM NaF, 1 mM PMSF, EDTA-free protease inhibitor cocktail). Cell lysates were snap frozen in liquid nitrogen, then boiled for 5 min and sonicated before centrifugation.
Cell lysates were separate by SDS-PAGE using pre-cast 4–12% Bis-Tris gels (Life Technologies). Proteins were transferred onto PVDF membranes (Immobilon-P; Millipore). Membranes were blocked in 5% skimmed milk powder 5% BSA, incubated with primary antibodies in blocking solution, followed by HRP-conjugated secondary antibodies and detection by enhanced chemiluminescence (ECL) (GE Healthcare) according to the manufacturer’s instructions. Bands on immunoblots were quantified by densitometric analysis using ImageJ software.
RhoA activity assay
GST-Rhotekin-RBD was purified from E. coli on glutathione sepharose beads (GE Healthcare) as previously described . HUVECs were lysed with Rho lysis buffer (50 mM Tris-Cl pH 7.5, 500 mM NaCl, 10 mM MgCl2, 10% glycerol, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 25 mM NaF, 1 mM Na3VO4, 1 mM PMSF, EDTA-free protease inhibitor cocktail). A small aliquot of the lysate was kept to determine total RhoA levels. Lysates were then incubated with GST-RBD for 1 h at 4 °C with rotation. Protein was eluted from the beads by boiling with 4× Laemmli sample buffer and analysed by western blotting.
Immunofluorescence and confocal microscopy
HUVECs were seeded onto glass coverslips coated with fibronectin (10 μg/ml at 37 °C for overnight). Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked in 3% BSA. Primary antibodies were diluted in 1% BSA in PBS. Fluorophore-conjugated secondary antibodies, DAPI and phalloidin were prepared in the same way as the primary antibodies. Coverslips were mounted onto glass slides using fluorescent mounting medium (DAKO).
A Zeiss LSM510 confocal laser-scanning microscope with an EC Plan-Neofluar 40×/1.30 Oil DIC M27 or a Plan-Apochromat 63×/1.40 Oil DIC M27, and ZEN software was used to take images of fluorescently stained cells. Images in each experiment were acquired using the same gain and offset settings.
Stress fibers were quantified by assigning a score to each cell based on the stress fiber content in the centre of the cell; 0 – few or no stress fibers, 1 – up to 50% of the cell centre contains stress fibers, 2–50% to 75% of the cell centre contains stress fibers, 3 – greater than 75% of the cell centre contains stress fibers. The experimenter quantifying stress fibers was blinded to the treatment.
Endothelial permeability assay
HUVECs were transfected with siRNAs and after 48 h were plated onto fibronectin-coated (10 μg/ml at 37 °C for 1 h) Transwell filters (12-mm diameter, 0.4-μm pore size, Costar) to form confluent monolayers. After 24 h, 0.1 mg/ml FITC dextran (molecular weight 42 kDa) was added to the upper chamber. Fluorescence was measured in the lower chamber after 80 min using a microplate analyser (Fusion-FA; PerkinElmer; excitation, 485 nm; detection, 523–535 nm). Each condition was performed in triplicate.
Angiogenic loop formation assay
Matrigel (BD Biosciences, at least 9 mg/ml) was diluted 1:1 with PBS, 300 μl added to each well of a 6-well dish and allowed to polymerize for 1.5 h. HUVECs were transfected with siRNAs and after 48 h 2 × 105 cells per well were seeded onto Matrigel, with or without addition of 10 μM ROCK inhibitor Y-27632 (Calbiochem). Cells were imaged after 24 h by phase-contrast microscopy using a Nikon TE2000-E microscope with a Plan Fluor 4× or 10× objective (Nikon) and a Hamamatsu Orca-ER digital camera, or fixed, permeabilized and stained for F-actin as described above (Immunofluorescence and confocal microscopy). The number of loops formed per imaged field was counted. The mean value of loops for multiple fields was used for statistical analysis. Alternatively, phase-contrast time-lapse movies were acquired 1 h after seeding cells onto Matrigel at 37 °C and 5% CO2. Images were captured using Metamorph software at a frame rate of 1 frame/30 min for 24 h.
Total RNA was extracted 72 h after siRNA transfection using either an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions or with Trizol and chloroform extraction. Purified RNA was treated with DNase (DNA free kit, Ambion). A SuperScript® VILO™ cDNA synthesis kit (Invitrogen) was used to synthesise cDNA according to the manufacturer’s instructions.
qPCR was carried out with SYBR green detection chemistry (mastermix from Primer Design). GAPDH was used as a reference gene. Data were acquired using either an ABI Prism 7000 system (Applied Biosystems) or the MX3005p system (Agilent Technologies) and analysed with ABI7000 SDS analysis software or with MxPro QPCR software respectively. Cycle to threshold (CT) values were determined for each condition, normalised to GAPDH levels and % mRNA expression normalised to control siRNA calculated as (100/(2 ^ (CT shift)).